Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high‐resolution membrane capacitance (Cm) measurements. The total whole‐cell Cm and the amplitudes and times of discrete femtoFarad (fF)‐sized Cm steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca2+]i was elevated by intracellular photolysis of calcium‐DM‐nitrophen to evoke secretion and monitored with the low‐affinity Ca2+ indicator furaptra. Sustained elevation of [Ca2+]i to > 20 μm evoked large, slow increases in Cm of up to 5 pF in 1‐2 min. Exocytotic and endocytotic steps of amplitude 0.5‐110 fF were resolved, and accounted on average for ≈33 % of the total Cm change. A prominent component of Cm steps of 2.5‐9.0 fF was seen and could be attributed to exocytosis of von‐Willebrand‐factor‐containing Weibel‐Palade bodies (WPb), based on the near‐identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5‐9.0 fF Cm steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca2+]i elevation, whereas total Cm increased immediately due to the secretion of small, non‐WPb granules. The results show that following a large increase of [Ca2+]i, corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9‐110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.

SUMMARY1. The changes in ciliary beat frequency (CBF) of human nasal respiratory epithelial cells were measured in vitro with a photometric technique following exposure to either 4-bromo-calcium ionophore A23187 (4-Br-A23187) or trifluoperazine (TFP), an inhibitor of calmodulin-sensitive calcium-dependent protein kinases. Changes in intracellular free calcium concentrations in response to 4-Br-A23187 were studied using a fluorescent dye (Fura-2).2. Addition of 10' M-4-Br-A23187 caused a time-dependent (P < 001) rise in CBF. The increment in CBF was statistically significant 10 min after challenge (+ 10 %; P < 0 01) and was sustained for at least 1 h, with maximal stimulation after 40 min (+ 18 %; P < 0-01).3. Exposure to 10' M-4-Br-A23187 caused an immediate increase in intracellular free calcium concentration, which preceded the rise in CBF.4. TFP (10' M) caused a reduction of baseline CBF (-10%; P < 001) and prevented the expected rise when the cells were subsequently exposed to 10' M-4-Br-A23187.5. We conclude that: (1) calcium ionophore stimulates the CBF of human respiratory cells; (2) this effect is mediated through a calmodulin-sensitive system, since it is abolished in the presence of TFP; (3) the same pathway appears to control the basal CBF of these cells, since TFP also decreases CBF.

Following irritation of the airway, the ciliostimulatory effects of the tachykinin, substance P (SP), are thought to be secondary to mucus release. We hypothesized that SP also induces small increases in ciliary beat frequency (CBF) via a calcium-mediated process.Brushed ciliated cells from the nasal epithelium of healthy human subjects were suspended in tissue culture fluid and the acute effects of SP upon these cells were studied in a mucus-free environment. In some preparations, changes in CBF in response to SP were measured with a video-based system. The effect of an SP antagonist, of Ca 2+ channel block with verapamil, and of the calcium analogue lanthanum on the SP response were also tested. In other preparations, the ciliated cells were preloaded with Fura-2, a dye which fluoresces with Ca 2+ ions, and the response of intracellular Ca 2+ to SP was monitored.SP ( We conclude that substance P induces either a transient or sustained increase in CBF dependent on the rate of destruction of this peptide around tachykinin receptors. These receptors are likely to be linked to lanthanum-and verapamil-sensitive pathways for the entry of Ca 2+ into cells. The small magnitude of the rise in CBF makes its physiological role uncertain at present.

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The culture of rat submandibular ganglion cells is described. The neurons can be distinguished from the non-neuronal cells in the cultures by their morphology. Recording with whole-cell voltage-clamp techniques indicates that the neurons have resting potentials of about – 55 mV and that the kinetics of the ionic channels opened by locally perfused acetylcholine (ACh) are very similar to those previously observed in adult submandibular ganglion neurons. The major differences observed are that the recorded cell input impedances are much higher than those recorded with microelectrodes from adult neurons and that the sensitivity of the cultured neurons to ACh is much less than that of the adult neurons. Whether the latter is due to changed receptor properties or to the presence of fewer receptors is not known.